The Inhibitory Effect Of Tissue Inhibitor Metalloproteinase-1 Mediated By Recombinant Adeno-associated Virus-2 On Hepatocellular Carcinoma Growth And Invasion In Vitro And In Vivo

Objective This study was design to conduct (a) to construct a recombinant adeno-associated virus-2 carrying tissue inhibitor metalloproteinase-l(TIMP1) gene (rAAV2-TIMP 1): (b) to study the effects that the rAAV2-TIMP 1 inhibit the invasiveness of hepatocellular carcinoma(HCC) cells in vitro model and the mchamism of rAAV2-TIMP1 in inhibiting the invasiveness ofHCC. (c) to study the effects on the growth and angiogenesis of HCC in vivo models by treatment of rAAV2-TIMP1. So as to investigate the possible potency of rAAV2-TIMP1 in treatment ofhepatocellular carcinomaMethods: Total RNA was extracted from human hepatocellular carcinoma tissue, and a 510-bp of TIMP 1 cDNA was amplified by RT-PCR and extracted, the TIMP 1 gene was then cloned into the adeno-assciated virus-2 vector pSNAV to form the recombinant pSNAV-TIMP1, Which was transfected into BHK-21 cells by means of lipofectamine. Using G418 Selection from mixed cells, BHK-TIMP1 was isolated, which was capable of TIMP1 expression and was subsequently infected with recombinant herpes simplex virus 1(HSVI-rc/AUL2) that was able to package the rAAV2-TIMP1 to form a functional and infectious virus. After purification, the packaged rAAV2-TIMP 1 was obtained. The human HCC cell line (Bel-7402) were transfected in vitro and treated in vivo with rAAV2-TIMP1, and a series of experiments was performed to observe the possoble effects of rAAV2-TIMP1 on the invasion and growth of Bel-7402 cells. Compared with the conventional transfection method, our modified method using "one provirus cell line, one helper virus" has an advantage to increase the yield of rAAV2 by two orders of magnitude. (a) The mRNA expression of TIMP 1 were detected by RT-PCR. (b) The protein production of TIMP 1 in the Bel-7402 cells was detected with Western Blot analysis. (c) The invasiveness of the Bel-7402 cells was assayed in Matrigel. (d) The index of proliferation and apoptosis of Bel-7402 cells were detected with MTT and FCM respectively. (e) The in vitro experiments was done by subcutaneously inoculation with rAAV2-TIMP1 infected BHK-21 cells in nude mice, and injected intratumorally into pre-existing tumors. The tumors were removed, sectioned, stained with H&E and immunohistochemical method for inspection.Results: The recombinant adeno-associated virus-2 vector carrying TIMP1 was constructed successfully, Using RT-PCR and Western Blot, the destination gene TIMP1 was detected. The recombinat viral titer was 1×10¹²v.g/ml. Compared with PBS and rAAV2-1uc(empty control) infected cells, the invasiveness of Bel-7402 cells in Matrigel assays with transfected rAAV2-TIMP1 resulted in a significantly reduction by 52%, and the tumor mass was also obviously reduced in nude mice with subcutaneously inoculation by rAAV2-TIMP1. Direct intratumoral injection of rAAV2-TIMP 1 into pre-existing tumors significantly inhibited the further growth of the HCC tumor, the tumor cells show necrosis and shrink, and the apoptosis rate also increased in Bel-7402 cells infected with rAAV2-TIMP 1Conclusion: (1)The constructed recombinant adeno-associated virus-2 with TIMP 1 (rAAV2-TIMP1) can express TIMP 1 and effectively inhibit the invasiveness of Bel-7402 cells of HCC in vitro. (2) rAAV2-TIMP1 can effectively promote the apoptosis and inhibit the growth of Bel-7402 cells in vivo; (3) rAAV2-TIMP1 can effectively attenuate the growth of HCC in vivo models, and demonstrat a therapeutic potential for HCC.